High-throughput screening of glucocorticoid-induced enhancer activity reveals mechanisms of stress-related psychiatric disorders.

Exposure to stressful life events increases the risk for psychiatric disorders. Mechanistic insight into the genetic factors moderating the impact of stress can increase our understanding of disease processes. Here, we test 3,662 single nucleotide polymorphisms (SNPs) from preselected expression quantitative trait loci in massively parallel reporter assays to identify genetic variants that modulate the activity of regulatory elements sensitive to glucocorticoids, important mediators of the stress response. Of the tested SNP sequences, 547 were located in glucocorticoid-responsive regulatory elements of which 233 showed allele-dependent activity. Transcripts regulated by these functional variants were enriched for those differentially expressed in psychiatric disorders in the postmortem brain. Phenome-wide Mendelian randomization analysis in 4,439 phenotypes revealed potentially causal associations specifically in neurobehavioral traits, including major depression and other psychiatric disorders. Finally, a functional gene score derived from these variants was significantly associated with differences in the physiological stress response, suggesting that these variants may alter disease risk by moderating the individual set point of the stress response.

High-throughput Sequencing to Identify Bacteremia in Children with Cancer.

Current methods to diagnose bacteremia are limited. In this pilot study of children with cancer presenting with fever, we determined the concordance between a novel high-throughput sequencing platform called BacCapSeq and blood culture. High-throughput sequencing had modest concordance with blood culture. Discordant organisms included those with both unlikely or potential clinical relevance.

Expression of Lineage Transcription Factors Identifies Differences in Transition States of Induced Human Oligodendrocyte Differentiation.

Oligodendrocytes (OLs) are critical for myelination and are implicated in several brain disorders. Directed differentiation of human-induced OLs (iOLs) from pluripotent stem cells can be achieved by forced expression of different combinations of the transcription factors SOX10 (S), OLIG2 (O), and NKX6.2 (N). Here, we applied quantitative image analysis and single-cell transcriptomics to compare different transcription factor (TF) combinations for their efficacy towards robust OL lineage conversion. Compared with S alone, the combination of SON increases the number of iOLs and generates iOLs with a more complex morphology and higher expression levels of myelin-marker genes. RNA velocity analysis of individual cells reveals that S generates a population of oligodendrocyte-precursor cells (OPCs) that appear to be more immature than those generated by SON and to display distinct molecular properties. Our work highlights that TFs for generating iOPCs or iOLs should be chosen depending on the intended application or research question, and that SON might be beneficial to study more mature iOLs while S might be better suited to investigate iOPC biology.

Assessing genome-wide dynamic changes in enhancer activity during early mESC differentiation by FAIRE-STARR-seq.

Embryonic stem cells (ESCs) can differentiate into any given cell type and therefore represent a versatile model to study the link between gene regulation and differentiation. To quantitatively assess the dynamics of enhancer activity during the early stages of murine ESC differentiation, we analyzed accessible genomic regions using STARR-seq, a massively parallel reporter assay. This resulted in a genome-wide quantitative map of active mESC enhancers, in pluripotency and during the early stages of differentiation. We find that only a minority of accessible regions is active and that such regions are enriched near promoters, characterized by specific chromatin marks, enriched for distinct sequence motifs, and modeling shows that active regions can be predicted from sequence alone. Regions that change their activity upon retinoic acid-induced differentiation are more prevalent at distal intergenic regions when compared to constitutively active enhancers. Further, analysis of differentially active enhancers verified the contribution of individual TF motifs toward activity and inducibility as well as their role in regulating endogenous genes. Notably, the activity of retinoic acid receptor alpha (RARα) occupied regions can either increase or decrease upon the addition of its ligand, retinoic acid, with the direction of the change correlating with spacing and orientation of the RARα consensus motif and the co-occurrence of additional sequence motifs. Together, our genome-wide enhancer activity map elucidates features associated with enhancer activity levels, identifies regulatory regions disregarded by computational prediction tools, and provides a resource for future studies into regulatory elements in mESCs.

CRUP: a comprehensive framework to predict condition-specific regulatory units.

We present the software Condition-specific Regulatory Units Prediction (CRUP) to infer from epigenetic marks a list of regulatory units consisting of dynamically changing enhancers with their target genes. The workflow consists of a novel pre-trained enhancer predictor that can be reliably applied across cell types and species, solely based on histone modification ChIP-seq data. Enhancers are subsequently assigned to different conditions and correlated with gene expression to derive regulatory units. We thoroughly test and then apply CRUP to a rheumatoid arthritis model, identifying enhancer-gene pairs comprising known disease genes as well as new candidate genes.

Pluripotency reprogramming by competent and incompetent POU factors uncovers temporal dependency for Oct4 and Sox2.

Oct4, along with Sox2 and Klf4 (SK), can induce pluripotency but structurally similar factors like Oct6 cannot. To decode why Oct4 has this unique ability, we compare Oct4-binding, accessibility patterns and transcriptional waves with Oct6 and an Oct4 mutant defective in the dimerization with Sox2 (Oct4defSox2). We find that initial silencing of the somatic program proceeds indistinguishably with or without Oct4. Oct6 mitigates the mesenchymal-to-epithelial transition and derails reprogramming. These effects are a consequence of differences in genome-wide binding, as the early binding profile of Oct4defSox2 resembles Oct4, whilst Oct6 does not bind pluripotency enhancers. Nevertheless, in the Oct6-SK condition many otherwise Oct4-bound locations become accessible but chromatin opening is compromised when Oct4defSox2 occupies these sites. We find that Sox2 predominantly facilitates chromatin opening, whilst Oct4 serves an accessory role. Formation of Oct4/Sox2 heterodimers is essential for pluripotency establishment; however, reliance on Oct4/Sox2 heterodimers declines during pluripotency maintenance.

Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements.

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

Bowel and Bladder Injury Repair and Follow-up After Gynecologic Surgery.

Bowel and bladder injuries are relatively rare, but there can be serious complications of both open and minimally invasive gynecologic procedures. As with most surgical complications, timely recognition is key in minimizing serious patient morbidity and mortality. Diagnosis of such injuries requires careful attention to surgical entry and dissection techniques and employment of adjuvant diagnostic modalities. Repair of bowel and bladder may be performed robotically, laparoscopically, or using laparotomy. Repair of these injuries requires knowledge of anatomic layers and suture materials and testing to ensure that intact and safe repair has been achieved. The participation of consultants is encouraged depending on the primary surgeon's skill and expertise. Postoperative care after bowel or bladder injury requires surveillance for complications including repair site leak, abscess, and fistula formation.

Surgeon Volume in Benign Gynecologic Surgery: Review of Outcomes, Impact on Training, and Ethical Contexts.

It is becoming increasingly clear that surgeon volume affects surgical outcomes. High-volume surgeons demonstrate reduced perioperative complications, shorter operative times, and reduced blood loss during multiple modalities of benign gynecologic surgery. Furthermore, high-volume surgeons consistently demonstrate higher rates of minimally invasive approaches, low rates of conversion to laparotomy, and lower per-procedure case costs. It is suggested that surgeons who have completed postresidency training have improved surgical outcomes, although these data are limited. Surgical exposure in obstetrics and gynecology residency is varied and does not consistently meet demonstrated surgical learning curves. Deficiencies in residency surgical training may be related to the volume-outcome relationship. We suggest reforming residency surgical training and tracking postresidency practice to provide optimal surgical care. Additionally, surgeons may have an ethical obligation to inform patients of their surgical volume and outcomes, with options for referrals if needed.

Genomic dissection of enhancers uncovers principles of combinatorial regulation and cell type-specific wiring of enhancer-promoter contacts.

Genomic binding of transcription factors, like the glucocorticoid receptor (GR), is linked to the regulation of genes. However, as we show here, GR binding is a poor predictor of GR-dependent gene regulation even when taking the 3D organization of the genome into account. To connect GR binding sites to the regulation of genes in the endogenous genomic context, we turned to genome editing. By deleting GR binding sites, individually or in combination, we uncovered how cooperative interactions between binding sites contribute to the regulation of genes. Specifically, for the GR target gene GILZ, we show that the simultaneous presence of a cluster of GR binding sites is required for the activity of an individual enhancer and that the GR-dependent regulation of GILZ depends on multiple GR-bound enhancers. Further, by deleting GR binding sites that are shared between different cell types, we show how cell type-specific genome organization and enhancer-blocking can result in cell type-specific wiring of promoter-enhancer contacts. This rewiring allows an individual GR binding site shared between different cell types to direct the expression of distinct transcripts and thereby contributes to the cell type-specific consequences of glucocorticoid signaling.

Laparoscopic myomectomy and morcellation: A review of techniques, outcomes, and practice guidelines.

Laparoscopic myomectomy is a minimally invasive surgical approach to treat symptomatic uterine fibroids in women wishing for a uterine-sparing procedure. With careful patient selection, these procedures are associated with favorable reproductive outcomes and low perioperative morbidity. Current available methods for specimen retrieval include power and hand morcellation. The 2014 FDA safety warnings regarding power morcellation arose from concerns about the spread of occult malignancy and prompted widespread use of containment systems that may limit spread of myometrial cells. Investigation into the clinical effects of laparoscopic myomectomy and uncontained morcellation on the prognosis and spread of occult leiomyosarcoma has yielded mixed results. Other complications of uncontained power morcellation exist, including the development of parasitic leiomyomas. The FDA safety warnings have greatly influenced trends in benign gynecologic surgery, and survey data reflect trends in providers' opinions of these trends. In conclusion, recommendations for the current practice of laparoscopic myomectomy and morcellation are reviewed.

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells.

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2.

New paradigms in the conservative surgical and interventional management of adenomyosis.

PURPOSE OF REVIEW: Adenomyosis is commonly diagnosed in women of reproductive age. Interest in conservative interventions has grown as more women desire fertility preservation or avoidance of hysterectomy. This review discusses surgical and interventional methods for treatment of symptomatic adenomyosis. The technique, evidence, and utility of each method are described. RECENT FINDINGS: Hysteroscopic ablative techniques are associated with lower morbidity than with hysterectomy but may result in an unacceptable risk of treatment failure. Surgical adenomyomectomy may provide good symptomatic improvement, especially when combined with preoperative gonadotropin-releasing hormone agonist treatment. Laparoscopic myometrial coagulation is associated with high rates of future pregnancy complications. Uterine artery ligation has limited value as an isolated approach but, coupled with other techniques, provides adequate therapeutic control. Bilateral uterine artery embolization may improve symptoms, without significantly compromising fertility. Focused ultrasonic surgical methods also show promise in alleviating symptoms without compromising reproductive outcomes. SUMMARY: A multitude of surgical and interventional options are available for young women with symptomatic adenomyosis. These treatment methods have unique associated risks and benefits, and may have varying impacts on long-term symptom control, fertility, and reproductive outcomes.

Trends in malpractice claims for obstetric and gynecologic procedures, 2005 through 2014.

BACKGROUND: Interest in medical malpractice and areas of medicolegal vulnerability for practicing obstetricians and gynecologists has grown substantially, and many providers report changing surgical practice out of fear of litigation. Furthermore, education on medical malpractice and risk management is lacking for obstetrics and gynecology trainees. Recent obstetric and gynecologic malpractice claims data are lacking. We report on recent trends in malpractice claims for obstetrics and gynecology procedures, and compare these trends to those of other medical specialties. OBJECTIVE: We sought to evaluate recent trends in malpractice claims for obstetrics and gynecology procedures and compare these to other medical specialties. STUDY DESIGN: A search was performed on all medicolegal claims data for obstetrics and gynecology procedures from Jan. 1, 2005, through Dec. 31, 2014, using the Physician Insurers' Association of America data-sharing project, which was created to identify medical professional liability trends. Data from 20 insurance carriers were reviewed based on a search using International Classification of Diseases, Ninth Revision codes and unique database-specific codes. RESULTS: Of the 10,915 total claims closed from 2005 through 2014, the majority (59.5%) were dropped, withdrawn, or dismissed. The average indemnity of the remaining paid claims (31.1%) was $423,250. The most frequently litigated procedure was operative procedures on the uterus; 27.8% of cases were paid with an average indemnity of $279,384. The procedure associated with the highest proportion of paid claims was vacuum extraction. The average indemnity for paid obstetrics and gynecology procedural claims was 27% higher than that for all medical specialties combined. Obstetrics and gynecology procedural claims had the second highest average indemnity payment and the fifth highest paid-to-closed ratio of all medical specialties. CONCLUSION: Litigation claims for obstetrics and gynecology procedures have higher average indemnity payments and higher paid-to-closed ratios than most other medical specialties. Claims most frequently relate to gynecologic surgery, but obstetric procedures are more expensive. Possible factors may include procedural experience and unique perioperative complications. We encourage efforts addressing procedures, litigation, and quality interventions to improve outcomes, mitigate risk, and potentially lower indemnity payments.

Inactivation of intergenic enhancers by EBNA3A initiates and maintains polycomb signatures across a chromatin domain encoding CXCL10 and CXCL9.

Epstein-Barr virus (EBV) causes a persistent infection in human B cells by establishing specific transcription programs to control B cell activation and differentiation. Transcriptional reprogramming of EBV infected B cells is predominantly driven by the action of EBV nuclear antigens, among them the transcriptional repressor EBNA3A. By comparing gene expression profiles of wt and EBNA3A negative EBV infected B cells, we have previously identified a broad array of cellular genes controlled by EBNA3A. We now find that genes repressed by EBNA3A in these cells are significantly enriched for the repressive histone mark H3K27me3, which is installed by Polycomb group (PcG) proteins. This PcG-controlled subset of genes also carries H3K27me3 marks in a variety of other tissues, suggesting that the commitment to PcG silencing is an intrinsic feature of these gene loci that can be used by EBNA3A. In addition, EBNA3A targets frequently reside in co-regulated gene clusters. To study the mechanism of gene repression by EBNA3A and to evaluate the relative contribution of PcG proteins during this process, we have selected the genomic neighbors CXCL10 and CXCL9 as a model for co-repressed and PcG-controlled genes. We show that EBNA3A binds to CBF1 occupied intergenic enhancers located between CXCL10 and CXCL9 and displaces the transactivator EBNA2. This impairs enhancer activity, resulting in a rapid transcriptional shut-down of both genes in a CBF1-dependent manner and initiation of a delayed gain of H3K27me3 marks covering an extended chromatin domain. H3K27me3 marks increase gradually and are maintained by EBNA3A. Our study provides direct evidence that repression by EBNA3A requires CBF1 and that EBNA3A and EBNA2 compete for access to CBF1 at identical genomic sites. Most importantly, our results demonstrate that transcriptional silencing by EBNA3A precedes the appearance of repressive PcG marks and indicate that both events are triggered by loss of enhancer activity.